Molecular Biology Reports

, Volume 38, Issue 3, pp 2059–2065 | Cite as

Molecular cloning, characterization and expression analysis of adenylosuccinate lyase gene in grass carp (Ctenopharyngodon idella)

  • Tian Yuan
  • Ji-Rui Gu
  • Wen-Bo Gu
  • Jiang Wu
  • Shao-Rong Ge
  • Heng Xu


Adenylosuccinate lyase (ADSL) is a bifunctional enzyme acting in de novo purine synthesis and purine nucleotide recycling. In the present study, we have constructed a grass carp (Ctenopharyngodon idella) intestinal cDNA library that has over 2.3 × 105 primary clones. An expressed sequence tag (EST) of grass carp adenylosuccinate lyase (gcADSL) gene was screened from this library. Both 5′-RACE and 3′-RACE were carried out in order to obtain the complete cDNA sequence, which contains a 1,446 bp open reading frame encoding 482 amino acids about 54.552 kDa. The deduced amino acid sequence shares high homology with its vertebrate counterparts, which shares 94% similarity with zebrafish, 81% with African clawed frog as well as chicken, 77% with human and 76% with mouse. This gcADSL genomic sequence, consisted of 13 exons and 12 introns, is 8,557 bp in size. Real-time quantitative PCR analysis revealed that the highest expression level of gcADSL was detected in muscle and the lowest in gill. In western blotting analysis, His6-tagged gcADSL protein expressed in Escherichia coli could be recognized not only by an anti-His6-tag monoclonal antibody but also by an anti-human ADSL polyclonal antibody, indicating immunological crossreactivity occurs between grass carp and human ADSL protein. 1,082 bp 5′-flanking region sequence was cloned and analyzed.


cDNA library Expressed sequence tag Rapid amplification of cDNA ends Western blotting Genome Walking Real-time PCR 



This work was financial supported by The Grass Carp Genome Project from Tongwei Group Co., Ltd.

Supplementary material

11033_2010_331_MOESM1_ESM.tif (4 mb)
Online Resource 1 The nucleotide and deduced amino acid sequences of ADSL cDNA from grass carp. The start codon ATG and the stop codon TGA are shaded. The amino acid sequence is shown under the nucleotide sequence in a single letter code. The distal polyadenylation signal sequence is boxed (TIFF 4094 kb)
11033_2010_331_MOESM2_ESM.tif (4 mb)
Online Resource 2 Alignment of the amino-acid sequences of adenylosuccinate lyase from six vertebrates. Identical amino acids are indicated with asterisks (*), and the similar residues with (.). Amino acid numbering for each sequence is shown to the right of the sequence line. The highly conserved signature for β-elimination reactions are boxed while two histidine residues for general acid-general base catalysis are shaded and also boxed. M24, I70, P98, R139, R188, K244, R301, D420 and R424 in grass carp are shaded while N393 and G436 are shaded and also underlined. (TIFF 4086 kb)
11033_2010_331_MOESM3_ESM.tif (690 kb)
Online Resource 3 Phylogenetic tree showing the relationships of ADSL between grass carp and other vertebrates. Full length amino acid sequences were aligned using the Clustal X program and the phylogenetic tree was constructed using the Neighbour-Joining algorithm in MEGA version 4.0. (TIFF 690 kb)
11033_2010_331_MOESM4_ESM.doc (32 kb)
Online Resource 4 Exon–intron organization of the Grass Carp ADSL gene. (DOC 32 kb)
11033_2010_331_MOESM5_ESM.tif (2.2 mb)
Online Resource 5 Sequence of the 5′-flanking region of grass carp ADSL gene. Promoter element sequences are underlined. The bent arrow indicates the position of 5′-end of the cDNA sequence reported here. The start codon is in bold type. (TIFF 2299 kb)
11033_2010_331_MOESM6_ESM.tif (3.5 mb)
Online Resource 6 Comparison of gene structure of gcADSL with zebrafish, mouse and human ADSL. Exons and introns are indicated by boxes and lines, respectively. The numbers above the boxes and below the lines indicate the lengths of exons and introns in base pairs, respectively. The black boxes represent 3′ and 5′ UTR. Sizes of exon 1 and 13 are without 5′ and 3′ UTR. (TIFF 3592 kb)


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Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Tian Yuan
    • 1
  • Ji-Rui Gu
    • 2
  • Wen-Bo Gu
    • 1
  • Jiang Wu
    • 2
  • Shao-Rong Ge
    • 1
  • Heng Xu
    • 1
  1. 1.School of Life SciencesSichuan UniversityChengduPeople’s Republic of China
  2. 2.Tongwei Technology CenterChengduPeople’s Republic of China

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